Dendritic cells loaded with killed breast cancer cells induce differentiation of tumor-specific cytotoxic T lymphocytes

Background

Early clinical trials, mostly in the setting of melanoma, have shown that dendritic cells (DCs) expressing tumor antigens induce some immune responses and some clinical responses. A major difficulty is the extension to other tumors, such as breast carcinoma, for which few defined tumor-associated antigens are available. We have demonstrated, using both prostate carcinoma and melanoma as model systems, that DCs loaded with killed allogeneic tumor cell lines can induce CD8⁺ T cells to differentiate into cytotoxic T lymphocytes (CTLs) specific for shared tumor antigens.

Methods

The present study was designed to determine whether DCs would capture killed breast cancer cells and present their antigens to autologous CD4⁺ and CD8⁺ T cells.

Results

We show that killed breast cancer cells are captured by immature DCs that, after induced maturation, can efficiently present MHC class I and class II peptides to CD8⁺ and CD4⁺ T lymphocytes. The elicited CTLs are able to kill the target cells without a need for pretreatment with interferon gamma. CTLs can be obtained by culturing the DCs loaded with killed breast cancer cells with unseparated peripheral blood lymphocytes, indicating that the DCs can overcome any potential inhibitory effects of breast cancer cells.

Conclusion

Loading DCs with killed breast cancer cells may be considered a novel approach to breast cancer immunotherapy and to identification of shared breast cancer antigens.

     

Chemotherapeutic agents in low noncytotoxic concentrations increase immunogenicity of human colon cancer cells

Background

We have recently reported that chemotherapeutic agents in ultra low noncytotoxic concentrations may block the ability of tumor cells to suppress functional activation of dendritic cells (DCs).

Methods

HCT-116 human colon cancer cells were treated with 0.5 nM paclitaxel (PAC) or 2 nM doxorubicin (DOX) with the aim of defining the immunogenic changes induced by ultra low noncytotoxic concentrations of antineoplastic chemotherapeutic agents. Genetic alterations were screened by DNA microarray that revealed increased expression of genes involved in antigen processing and presentation, including the heat-shock protein, calmodulin, and proteasome 26 genes. As the proteins encoded by these genes are involved in the cytosolic route of antigen processing machinery, we next evaluated whether PAC and DOX in noncytotoxic concentrations changed expression of MHC class I antigen processing machinery (APM) components in three different colon cancer cell lines.

Results

Our results showed that PAC and DOX increased the intracellular expression of APM proteins, including calmodulin, LMP2, LMP7, TAP1 and tapasin. The biological significance of modulation of antigen processing and presentation proteins in tumor cells by ultra low nontoxic concentrations of chemotherapeutic drugs was revealed when non-treated and treated tumor cells were used as a source of tumor antigens for the generation of tumor-specific cytotoxic T cells (CTLs) in vitro. We demonstrated that (i) DCs that engulf tumor cells pretreated with noncytotoxic concentrations of chemotherapeutic agents induced CTLs with a higher cytotoxic potential than DCs loaded with nontreated tumor cells, and (ii) CTLs induced by tumor lysate-pulsed DCs killed live tumor cells more efficiently if these tumor cells were pretreated with noncytotoxic concentrations of chemotherapeutic drugs.

Conclusions

These results demonstrate that chemomodulation of human tumor cells with noncytotoxic concentrations of chemotherapeutic agents increases tumor immunogenicity and results in the generation of more efficient DC vaccines and CTLs, which can be used for cell-based anticancer immunotherapies.     

Targeting of the non-mutated tumor antigen HER2/neu to mature dendritic cells induces an integrated immune response that protects against breast cancer in mice

Introduction

Given their relative simplicity of manufacture and ability to be injected repeatedly, vaccines in a protein format are attractive for breast and other cancers. However, soluble human epidermal growth factor receptor (HER2)/neu protein as a vaccine has not been immunogenic. When protein is directly targeted to antigen uptake receptors, such as DEC205 (DEC), efficient processing and presentation of antigen take place. The aim of this study was to determine the immunogenicity of a HER2 protein vaccine that directly targets to DEC⁺ dendritic cells (DCs) in a mouse breast cancer model.

Methods

We genetically engineered the HER2 extracellular domain into a monoclonal antibody specific for DEC (DEC-HER2). Mice of various genetic backgrounds were immunized with DEC-HER2 in combination with DC maturation stimuli (poly IC ± CD40 Ab). Vaccine-induced T cell immunity was determined by analyzing the ability of CD4⁺/CD8⁺ T cell to produce interferon (IFN)-gamma and proliferate upon antigen rechallenge. Sera were assessed for the presence of antigen specific antibody (Ab). For vaccine efficacy, FVB/N mice were immunized with DEC-HER2 in combination with poly IC and protection against neu-expressing mammary tumors was assessed. Protection mechanisms and tumor-specific T cell responses were also evaluated.

Results

We demonstrate that DEC-HER2 fusion mAb, but not Ctrl Ig-HER2, elicits strong, broad and multifunctional CD4⁺ T cell immunity, CD8⁺ T cell responses, and humoral immunity specific for HER2 antigen. Cross-reactivity to rat neu protein was also observed. Importantly, mice xeno-primed with DEC-HER2 were protected from a neu-expressing mammary tumor challenge. Both CD4⁺ and CD8⁺ T cells mediated the tumor protection. Robust anti-tumor T cell immunity was detected in tumor protected mice.

Conclusions

Immunization of mice with HER2 protein vaccine targeting DEC⁺ DCs in vivo induced high levels of T- and B-cell immunity. Non-targeted HER2 protein was poorly immunogenic for CD4⁺ and CD8⁺ T cells. This vaccination approach provided long-term survival benefit for mice challenged with neu-expressing tumor following as little as 2.7 ??g of HER2 protein incorporated in the vaccine. Vaccine-induced CD4⁺ and CD8⁺ T cells were both essential for tumor protection. This immunization strategy demonstrates great potential towards the development of vaccines for breast cancer patients.     

Generation and characterization of GP-100 peptide-specific NK-T cell clones

MHC-restricted cytotoxic T lymphocytes (CTLs) specific for antigens expressed by malignant cells are important components of immune responses against human cancer. Peripheral blood monocytes of HLA-A2⁺ healthy donors were used to induce dendritic cells (DCs) by granulocyte-macrophage colony-stimulating factor and interleukin-4 and loaded with a gp100 peptide (YLEPGPVTA). By applying these peptide-loaded DCs, a CTL line that displayed high cytotoxic reactivity with peptide-loaded target cells was generated. A total of 11 gp100 peptide-specific CTL clones were generated from this cell line. Several of these CTL clones were studied in detail. Of particular interest was clone CTL-45, which, contrary to the parental cell line, displayed strong NK activity and, by flow-cytometric analysis, revealed a CD3⁺, TCR BV17, CD8⁺ and CD56⁺ phenotype. This clone was strictly peptide-specific and effectively killed a panel of melanoma cells expressing HLA-A2 and gp100. Tumor-specific T cells with this kind of dual function are potentially of great clinical importance as they have a backup mechanism that may go into action when tumor cells escape specific killing by losing their HLA-class I molecules. Int. J. Cancer 75:794–803, 1998.© 1998 Wiley-Liss, Inc.     

A different immunologic profile characterizes patients with HER-2-overexpressing and HER-2-negative locally advanced breast cancer: implications for immune-based therapies

Introduction

The clinical efficacy of trastuzumab and taxanes is at least partly related to their ability to mediate or promote antitumor immune responses. On these grounds, a careful analysis of basal immune profile may be capital to dissect the heterogeneity of clinical responses to these drugs in patients with locally advanced breast cancer undergoing neoadjuvant chemotherapy.

Methods

Blood samples were collected from 61 locally advanced breast cancers (36 HER2^- and 25 HER2⁺) at diagnosis and from 23 healthy women. Immunophenotypic profiling of circulating and intratumor immune cells, including regulatory T (Treg) cells, was assessed by flow cytometry and immunohistochemistry, respectively. Serum levels of 10 different cytokines were assessed by multiplex immunoassays. CD8⁺ T cell responses to multiple tumor-associated antigens (TAA) were evaluated by IFN-γ-enzyme-linked immunosorbent spot (ELISPOT). The Student's t test for two tailed distributions and the Wilcoxon two-sample test were used for the statistical analysis of the data.

Results

The proportion of circulating immune effectors was similar in HER2⁺ patients and healthy donors, whereas higher percentages of natural killer and Treg cells and a lower CD4⁺/CD8⁺ T cell ratio (with a prevalence of naïve and central memory CD8⁺ T cells) were observed in HER2^- cases. Higher numbers of circulating CD8⁺ T cells specific for several HLA-A*0201-restricted TAA-derived peptides were observed in HER2⁺ cases, together with a higher prevalence of intratumor CD8⁺ T cells. Serum cytokine profile of HER2⁺ patients was similar to that of controls, whereas HER2^- cases showed significantly lower cytokine amounts compared to healthy women (IL-2, IL-8, IL-6) and HER2⁺ cases (IL-2, IL-1β, IL-8, IL-6, IL-10).

Conclusions

Compared to HER2- cases, patients with HER2-overexpressing locally advanced breast cancer show a more limited tumor-related immune suppression. This may account for the clinical benefit achieved in this subset of patients with the use of drugs acting through, but also promoting, immune-mediated effects.     

Human breast cancer cell lines contain stem-like cells that self-renew, give rise to phenotypically diverse progeny and survive chemotherapy

Introduction

The phenotypic and functional differences between cells that initiate human breast tumors (cancer stem cells) and those that comprise the tumor bulk are difficult to study using only primary tumor tissue. We embarked on this study hypothesizing that breast cancer cell lines would contain analogous hierarchical differentiation programs to those found in primary breast tumors.

Methods

Eight human breast cell lines (human mammary epithelial cells, and MCF10A, MCF7, SUM149, SUM159, SUM1315 and MDA.MB.231 cells) were analyzed using flow cytometry for CD44, CD24, and epithelial-specific antigen (ESA) expression. Limiting dilution orthotopic injections were used to evaluate tumor initiation, while serial colony-forming unit, reconstitution and tumorsphere assays were performed to assess self-renewal and differentiation. Pulse-chase bromodeoxyuridine (5-bromo-2-deoxyuridine [BrdU]) labeling was used to examine cell cycle and label-retention of cancer stem cells. Cells were treated with paclitaxol and 5-fluorouracil to test selective resistance to chemotherapy, and gene expression profile after chemotherapy were examined.

Results

The percentage of CD44⁺/CD24^- cells within cell lines does not correlate with tumorigenicity, but as few as 100 cells can form tumors when sorted for CD44⁺/CD24^-/low/ESA⁺. Furthermore, CD44⁺/CD24^-/ESA⁺ cells can self-renew, reconstitute the parental cell line, retain BrdU label, and preferentially survive chemotherapy.

Conclusion

These data validate the use of cancer cell lines as models for the development and testing of novel therapeutics aimed at eradicating cancer stem cells.     

CD44+/CD24− breast cancer cells isolated from MCF-7 cultures exhibit enhanced angiogenic properties

Background

Recent studies suggest that the relationship between cancer stem cells (CSCs) and the vascular niche may be bidirectional; the niche can support the growth and renewal of CSCs, and CSCs may contribute to the maintenance of the niche. There is little knowledge concerning the role of breast cancer stem cells in promoting tumor angiogenesis.

Aim

For human breast cancers, CSCs have been shown to be associated with a CD44⁺/CD24 ^? phenotype. We investigated the potential activities of CD44⁺/CD24 ^? breast cancer stem cells in promoting tumor angiogenesis.

Methods

The expression of pro-angiogenic genes was determined by quantitative real-time RT-PCR. Endothelial cell migration assays were employed to evaluate effects of conditioned media from CD44⁺/CD24 ^? on human umbilical vein endothelial cells. A chorioallantoic membrane (CAM) assay was used to study the potential of CD44⁺/CD24 ^? cells to promote angiogenesis.

Results

In our study, CD44⁺/CD24 ^? cells expressed elevated levels of pro-angiogenic factors compared with CD44⁺/CD24⁺ cells. CD44⁺/CD24 ^? cell-conditioned media significantly increased endothelial cell migration. Breast cancer cell lines enriched with CD44⁺/CD24 ^? cells were more pro-angiogenic in the CAM assay than those lacking a CD44⁺/CD24 ^? subpopulation. CD44⁺/CD24 ^? cells sorted from MCF-7 cell lines were more pro-angiogenic in a CAM assay than CD44⁺/CD24⁺ cells. Furthermore, the VEGF concentration was significantly higher in CD44⁺/CD24 ^? cell-conditioned media than in CD44⁺/CD24⁺ cell-conditioned media. The pro-angiogenic effect of CD44⁺/CD24 ^? cells on endothelial cells was abolished by bevacizumab.

Conclusion

Our findings demonstrate that CD44⁺/CD24 ^? breast cancer stem cells have substantial pro-angiogenic potential and activity. This provides new insights to explore in the development of targeted therapies.     

Additive growth inhibitory effects of ibandronate and antiestrogens in estrogen receptor-positive breast cancer cell lines

Introduction

Dendritic cells (DCs) are key antigen-presenting cells that play an essential role in initiating and directing cellular and humoral immunity, including anti-tumor responses. Due to their critical role in cancer, induction of DC apoptosis may be one of the central mechanisms used by tumors to evade immune recognition.

Methods

Spontaneous apoptosis of blood DCs (lineage negative HLA-DR positive cells) was assessed in peripheral blood mononuclear cells (PBMCs) using Annexin-V and TUNEL assays immediately after blood collection. The role of tumor products was assessed by culturing cells with supernatants derived from breast cancer cell lines (TDSN) or PBMCs (PBMC-SN, as a control). The capacity of DC stimulation to prevent apoptosis was assessed by incubating DC with inflammatory cytokines, poly I:C, IL-12 or CD40 ligand (CD40L) prior to culture with TDSN. Apoptosis was determined by flow cytometry and microscopy, and Bcl-2 expression determined by intracellular staining.

Results

In this study we document the presence of a significantly higher proportion of apoptotic (Annexin-V⁺ and TUNEL⁺) blood DCs in patients with early stage breast cancer (stage I to II; n = 13) compared to healthy volunteers (n = 15). We examined the role of tumor products in this phenomenon and show that supernatants derived from breast cancer lines induce apoptosis of blood DCs in PBMC cultures. Aiming to identify factors that protect blood DC from apoptosis, we compared a range of clinically available maturation stimuli, including inflammatory cytokines (tumor necrosis factor-α, IL-1β, IL-6 and prostaglandin (PG)E₂ as a cytokine cocktail), synthetic double-stranded RNA (poly I:C) and soluble CD40 ligand. Although inflammatory cytokines and poly I:C induced robust phenotypic maturation, they failed to protect blood DCs from apoptosis. In contrast, CD40 stimulation induced strong antigen uptake, secretion of IL-12 and protected blood DCs from apoptosis through sustained expression of Bcl-2. Exogenous IL-12 provided similar Bcl-2 mediated protection, suggesting that CD40L effect is mediated, at least in part, through IL-12 secretion.

Conclusion

Cumulatively, our results demonstrate spontaneous apoptosis of blood DCs in patients with breast cancer and confirm that ex vivo conditioning of blood DCs can protect them from tumor-induced apoptosis.     

Functional heterogeneity of circulating T regulatory cell subsets in breast cancer patients

Background

Regulatory T cells (Tregs) play a major role in tumor escape from immunosurveillance by suppressing effector cells. The number of Tregs is increased in tumor sites and peripheral blood of breast cancer patients. However, the data regarding phenotypic and functional heterogeneity of Treg subpopulations in breast cancer are limited. The present study aimed to investigate the number and suppressive potential of Tregs that possess natural naïve-(N nTregs), effector/memory-like (EM nTregs), and Tr1-like phenotypes in breast cancer patients and healthy women.

Methods

The study included 10 HW and 17 primary breast cancer patients. Numbers of CD4⁺CD25⁺FoxP3⁺CD45RA⁺ N nTregs, CD4⁺CD25⁺FoxP3⁺CD45RA^? EM nTregs, and CD4⁺IL-4^?IL-10⁺ Tr1 subsets and the expression of CTLA-4, CD39, GITR, LAP, and IL-35 by these Treg subsets were measured in freshly obtained peripheral blood by flow cytometry.

Results

Herein, we demonstrate that the percentages of N nTregs, EM nTregs, CD25⁺ and FoxP3⁺ Tr1 cells are elevated in the peripheral blood of breast cancer patients, but do not correlate with cancer stages. Nevertheless, the frequency of CD25⁺ Tr1 cells was associated with nodal involvement, while the number of EM nTregs correlated with clinical outcome. The expression of CTLA-4 and IL-35 by all assessed Treg subsets was increased throughout all tumor stages (I–III).

Conclusions

Collectively, the current study shows phenotypic alterations in suppressive receptors of Treg subsets, suggesting that breast cancer patients have increased activity of N nTregs, EM nTregs and Tr1 cells; and EM nTregs and CD25⁺ Tr1 cells represent prospective markers for assessing disease prognosis.

     

Brca1 breast tumors contain distinct CD44+/CD24- and CD133+ cells with cancer stem cell characteristics

Introduction

Whether cancer stem cells occur in BRCA1-associated breast cancer and contribute to therapeutic response is not known.

Methods

We generated and characterized 16 cell lines from five distinct Brca1deficient mouse mammary tumors with respect to their cancer stem cell characteristics.

Results

All cell lines derived from one tumor included increased numbers of CD44⁺/CD24^- cells, which were previously identified as human breast cancer stem cells. All cell lines derived from another mammary tumor exhibited low levels of CD44⁺/CD24^- cells, but they harbored 2% to 5.9% CD133⁺ cells, which were previously associated with cancer stem cells in other human and murine tumors. When plated in the absence of attachment without presorting, only those cell lines that were enriched in either stem cell marker formed spheroids, which were further enriched in cells expressing the respective cancer stem cell marker. In contrast, cells sorted for CD44⁺/CD24^- or CD133⁺ markers lost their stem cell phenotype when cultured in monolayers. As few as 50 to 100 CD44⁺/CD24^- or CD133⁺ sorted cells rapidly formed tumors in nonobese diabetic/severe combined immunodeficient mice, whereas 50-fold to 100-fold higher numbers of parental or stem cell depleted cells were required to form few, slow-growing tumors. Expression of stem cell associated genes, including Oct4, Notch1, Aldh1, Fgfr1, and Sox1, was increased in CD44⁺/CD24^- and CD133⁺ cells. In addition, cells sorted for cancer stem cell markers and spheroid-forming cells were significantly more resistant to DNA-damaging drugs than were parental or stem cell depleted populations, and they were sensitized to the drugs by the heat shock protein-90 inhibitor 17-DMAG (17-dimethylaminoethylamino-17-demethoxygeldanamycin hydrochloride).

Conclusion

Brca1-deficient mouse mammary tumors harbor heterogeneous cancer stem cell populations, and CD44⁺/CD24^- cells represent a population that correlates with human breast cancer stem cells.     

Silencing IDO in dendritic cells: A novel approach to enhance cancer immunotherapy in a murine breast cancer model

Cancer immunotherapeutic agents (vaccines) in the form of antigen‐loaded dendritic cells (DCs) reached an important milestone with the recent approval of Provenge, the first DC vaccine for treatment of prostate cancer. Although this heralds a new era of tumor immunotherapy, it also highlights the compelling need to optimize such DC‐based therapies as they are increasingly tested and used to treat human patients. In this study we sought to augment and enhance the antitumor activity of a DC‐based vaccine using siRNA to silence expression of immunosuppressive enzyme indoleamine 2,3‐dioxygenase (IDO) in DCs. We report here that DCs loaded with tumor antigens, but with siRNA‐silenced IDO expression, were introduced into 4T1 breast tumor‐bearing mice, the treatment: (i) lengthened the time required for tumor onset, (ii) decreased tumor size compared to tumors grown for equal lengths of time in mice treated with antigen‐loaded DCs without IDO silencing and (iii) reduced CD4⁺ and CD8⁺ T cell apoptosis. Furthermore, immunization with IDO‐silenced DCs enhanced tumor antigen‐specific T cell proliferation and CTL activity, and decreased numbers of CD4⁺CD25⁺Foxp3⁺ T_reg. This study provides evidence to support silencing of immunosuppressive genes (IDO) as an effective strategy to enhance the efficacy of DC‐based cancer immunotherapeutic.     

The clinical significance of memory T cells and its subsets in gastric cancer

Background

Long life of memory T cell (Tm) determines its crucial role in the carcinogenesis and carcinogenic progression which usually take long time. The Tm compartment contains two populations, central memory T cells (Tcm) and effector memory T cells (Tem), based on their phenotypic markers, functional attributes, and migratory properties.

Methods

We investigated the subsets of the Tm in peripheral blood and tumor microenvironments in patients with gastric cancer by flow cytometry, and aimed to explore the correlation between the Tm and clinicopathologic features of gastric cancer.

Results

The percentages of CD4⁺/CD8⁺ Tm and CD4⁺/CD8⁺ Tcm in peripheral blood from gastric cancer patients were statistically lower, whereas the percentages of CD4⁺/CD8⁺ Tem were significantly higher than healthy controls. The proportion of CD4⁺/CD8⁺ Tcm increased after tumor resection, while the percentage of the CD4⁺/CD8⁺ Tem decreased significantly. Significant associations were detected between the peripheral CD4⁺/CD8⁺ Tm and clinical stage, as well as the CD8⁺ Tcm and clinical stage and nodal involvement. Tumor infiltrating CD8⁺ Tm expressed both central and effector memory phenotypes, whereas CD4⁺ Tm displayed predominantly an effector memory phenotype. Higher percentages of tumor infiltrating CD4⁺/CD8⁺ Tm were significantly associated with the early disease stage.

Conclusions

Tm and its subsets were good immune indicators for the disease stage of gastric cancer. The proportion of Tm subsets may reflect the immune suppressive and immune response to the tumor associated antigen.     

Deoxycholate promotes survival of breast cancer cells by reducing the level of pro-apoptotic ceramide

Introduction

At physiologic concentration in serum, the bile acid sodium deoxycholate (DC) induces survival and migration of breast cancer cells. Here we provide evidence of a novel mechanism by which DC reduces apoptosis that is induced by the sphingolipid ceramide in breast cancer cells.

Methods

Murine mammacarcinoma 4T1 cells were used in vitro to determine apoptosis and alteration of sphingolipid metabolism by DC, and in vivo to quantify the effect of DC on metastasis.

Results

We found that DC increased the number of intestinal metastases generated from 4T1 cell tumors grafted into the fat pad. The metastatic nodes contained slowly dividing cancer cells in immediate vicinity of newly formed blood vessels. These cells were positive for CD44, a marker that has been suggested to be expressed on breast cancer stem cells. In culture, a subpopulation (3 ± 1%) of slowly dividing, CD44⁺ cells gave rise to rapidly dividing, CD44^- cells. DC promoted survival of CD44⁺ cells, which was concurrent with reduced levels of activated caspase 3 and ceramide, a sphingolipid inducing apoptosis in 4T1 cells. Z-guggulsterone, an antagonist of the farnesoid-X-receptor, obliterated this anti-apoptotic effect, indicating that DC increased cell survival via farnesoid-X-receptor. DC also increased the gene expression of the vascular endothelial growth factor receptor 2 (Flk-1), suggesting that DC enhanced the initial growth of secondary tumors adjacent to blood vessels. The Flk-1 antagonist SU5416 obliterated the reduction of ceramide and apoptosis by DC, indicating that enhanced cell survival is due to Flk-1-induced reduction in ceramide.

Conclusions

Our findings show, for the first time, that DC is a natural tumor promoter by elevating Flk-1 and decreasing ceramide-mediated apoptosis of breast cancer progenitor cells. Reducing the level or effect of serum DC and elevating ceramide in breast cancer progenitor cells by treatment with Z-guggulsterone and/or vascular endothelial growth factor receptor 2/Flk-1 antagonists may thus be a promising strategy to reduce breast cancer metastasis.     

Pharmacological inhibition of AKT sensitizes MCF-7 human breast cancer-initiating cells to radiation

Background

Caner-initiating cells (CICs or cancer stem cells) have been shown both experimentally and clinically to be resistant to radiation. The mechanism underlying radioresistance remains unclear.

Methods

In the present study, we screened 51 genes which are potentially important in mediating radioresistance of breast CICs.

Results

The expression of AKT1 and AKT2 at protein and mRNA levels was dramatically increased among the screened genes by 8 Gy radiation treatment in MCF-7 mammosphere cells (predominantly CD24^–/low/CD44⁺ CICs), but not in the bulk population of MCF-7 cells (predominantly CD24⁺/CD44⁺). Using apoptosis and clonogenic survival assays, we found pharmacological inhibition of AKT with selective inhibitors of AKT sensitized MCF-7 mammosphere cells, but not MCF-7 monolayer cells to radiation.

Conclusion

The present findings suggest that treatment with AKT inhibitors prior to ionizing radiation treatment may be a potential benefit to patients with breast cancer, in particular to eradiate breast CICs.     

Dendritic cells transfected with hepatocellular carcinoma (HCC) total RNA induce specific immune responses against HCC in vitro and in vivo

Background

Immunotherapy is an effective method for preventing metastasis and recurrence of carcinoma. Hepatocellular carcinoma (HCC) is a common malignancy with a high rate of recurrence, and has not successfully been introduced to immunotherapy.

Methods

Peripheral blood mononuclear cells were isolated from whole blood of HCC patients and stimulated to transform into dendritic cells (DCs). These DCs were then transfected with RNA extracted from HepG-2 hepatoma cells to induce expression of specific antigens.

Results

The transfected DCs stimulated T lymphocytes to produce cytotoxic T lymphocytes, which specifically attacked HepG-2 cells. Injection of T lymphocytes from HCC patients and transfected DCs into severe combined immunodeficiency mice limited the growth of HepG-2 tumors.

Conclusion

A specific immune response against hepatoma can be generated in vivo by administering DCs transfected with RNA from a specific tumor. This method may have therapeutic application in humans to reduce recurrence of HCC.     

Influence of ovarian cancer type I and type II microenvironment on the phenotype and function of monocyte-derived dendritic cells

PURPOSE

The aim of this study was to evaluate the influence of ovarian cancer cell lysates isolated from type I or type II ovarian cancer (OC) on the phenotype of monocyte-derived dendritic cells (Mo-DCs) and the cytokine profile. We also determined whether the Mo-DCs and tumor microenvironment, reflected by peritoneal fluid (PF) from type I or II ovarian cancer, could promote regulatory T cell (Tregs) differentiation from naive CD4⁺ lymphocytes in vitro.

RESULTS

Our results show a significant role of the ovarian cancer microenvironment reflected by PF from type I or II OC in the inhibition of the DC differentiation process. Interestingly, the percentage of cells co-expressing CD45 and CD14 antigens in the cultures stimulated with PF from both type I and type II OC was higher than in the control. Furthermore, the percentage of cells expressing CD1a, i.e., a marker of immature DCs, was significantly reduced in the cultures stimulated with PF from type I and type II OC. The results obtained show that ovarian cancer type II lysates induce differentiation of monocytes into macrophage-like cells with a CD1a⁺/HLA-DR⁺/CD83^? phenotype and significantly higher CD86/HLA-DR expression. We show that ovarian cancer type II Mo-DCs are able to prevent an immune response by release of IL-10, whereas OC type I Mo-DCs can promote the generation of Tregs.

CONCLUSIONS

We demonstrate that each type of ovarian cancer can induce a unique phenotype of DCs and differentiation of Tregs, both associated with immune-suppressive function, which may be an obstacle while developing effective anticancer dendritic cell vaccination.

     

Small interfering RNA library screen identified polo-like kinase-1 (PLK1) as a potential therapeutic target for breast cancer that uniquely eliminates tumor-initiating cells

Introduction

Triple-negative breast cancer (TNBC) high rate of relapse is thought to be due to the presence of tumor-initiating cells (TICs), molecularly defined as being CD44^high/CD24^-/low. TICs are resilient to chemotherapy and radiation. However, no currently accepted molecular target exists against TNBC and, moreover, TICs. Therefore, we sought the identification of kinase targets that inhibit TNBC growth and eliminate TICs.

Methods

A genome-wide human kinase small interfering RNA (siRNA) library (691 kinases) was screened against the TNBC cell line SUM149 for growth inhibition. Selected siRNAs were then tested on four different breast cancer cell lines to confirm the spectrum of activity. Their effect on the CD44^high subpopulation and sorted CD44^high/CD24^-/low cells of SUM149 also was studied. Further studies were focused on polo-like kinase 1 (PLK1), including its expression in breast cancer cell lines, effect on the CD44^high/CD24^-/low TIC subpopulation, growth inhibition, mammosphere formation, and apoptosis, as well as the activity of the PLK1 inhibitor, BI 2536.

Results

Of the 85 kinases identified in the screen, 28 of them were further silenced by siRNAs on MDA-MB-231 (TNBC), BT474-M1 (ER⁺/HER2⁺, a metastatic variant), and HR5 (ER⁺/HER2⁺, a trastuzumab-resistant model) cells and showed a broad spectrum of growth inhibition. Importantly, 12 of 28 kinases also reduced the CD44^high subpopulation compared with control in SUM149. Further tests of these 12 kinases directly on a sorted CD44^high/CD24^-/low TIC subpopulation of SUM149 cells confirmed their effect. Blocking PLK1 had the greatest growth inhibition on breast cancer cells and TICs by about 80% to 90% after 72 hours. PLK1 was universally expressed in breast cancer cell lines, representing all of the breast cancer subtypes, and was positively correlated to CD44. The PLK1 inhibitor BI 2536 showed similar effects on growth, mammosphere formation, and apoptosis as did PLK1 siRNAs. Finally, whereas paclitaxel, doxorubicin, and 5-fluorouracil enriched the CD44^high/CD24^-/low population compared with control in SUM149, subsequent treatment with BI 2536 killed the emergent population, suggesting that it could potentially be used to prevent relapse.

Conclusion

Inhibiting PLK1 with siRNA or BI 2536 blocked growth of TNBCs including the CD44^high/CD24^-/low TIC subpopulation and mammosphere formation. Thus, PLK1 could be a potential therapeutic target for the treatment of TNBC as well as other subtypes of breast cancer.     

Blood monocytes sample MelanA/MART1 antigen for long‐lasting cross‐presentation to CD8+ T cells after differentiation into dendritic cells

Human blood monocytes are very potent to take up antigens. Like macrophages in tissue, they efficiently degrade exogenous protein and are less efficient than dendritic cells (DCs) at cross‐presenting antigens to CD8⁺ T cells. Although it is generally accepted that DCs take up tissue antigens and then migrate to lymph nodes to prime T cells, the mechanisms of presentation of antigens taken up by monocytes are poorly documented so far. In the present work, we show that monocytes loaded in vitro with MelanA long peptides retain the capacity to stimulate antigen‐specific CD8⁺ T cell clones after 5 days of differentiation into monocytes‐derived dendritic cells (MoDCs). Tagged‐long peptides can be visualized in electron‐dense endocytic compartments distinct from lysosomes, suggesting that antigens can be protected from degradation for extended periods of time. To address the pathophysiological relevance of these findings, we screened blood monocytes from 18 metastatic melanoma patients and found that CD14⁺ monocytes from two patients effectively activate a MelanA‐specific CD8 T cell clone after in vitro differentiation into MoDCs. This in vivo sampling of tumor antigen by circulating monocytes might alter the tumor‐specific immune response and should be taken into account for cancer immunotherapy.     

CD80 expression in an HLA-A2-positive human non-small cell lung cancer cell line enhances tumor-specific cytotoxicity of HLA-A2-positive T cells derived from a normal donor and a patient with non-small cell lung cancer

To generate non-small cell lung cancer (NSCLC)–reactive lymphocytes, we transfected an HLA-A2-expressing human NSCLC line (1355) with the cDNA encoding the lymphocyte co-stimulatory molecule CD80. Following selection in G418, 1355.7 demonstrated stable cell-surface expression of CD80. Allogeneic mixed lymphocyte tumor cell cultures (MLTCs) were established in 600 IU/ml IL-2 using HLA-A2⁺ normal donor peripheral blood mononuclear cells (PBMCs) stimulated with 1355-P (parental), 1355.7 or IL-2 alone. In 7 of 9 MLTCs, those stimulated with 1355.7 demonstrated enhanced growth after 30 to 45 days of culture. The predominant lymphocyte to grow in all MLTCs was a CD3⁺αβ⁺CD4⁺ T cell. In one case, lymphocytes stimulated with 1355.7 (MLTC 2389.7) exhibited preferential lysis of 1355. MLTC 2389.7 was cloned by limiting dilution, and 2 resultant cloids were shown to be NSCLC-reactive and dependent on both MHC class I and CD3 in their recognition of tumor cells. Additionally, allogeneic MLTCs were established using three HLA-A2⁺ NSCLC patients' PBMC. The predominant lymphocyte to grow in these MLTCs was a CD3⁺ αβ⁺CD8⁺ T cell. In cytotoxicity studies, MLTC-UKY25.7 demonstrated preferential lysis of 1355-P, 1355.7 and an HLA-A2⁺ NSCLC cell line, 1650. Lymphocytes from this MLTC did not lyse K562, Daudi or an HLA-A2^– NSCLC cell line, 647. Our data suggest that CD80-expressing NSCLC tumor cells may enhance the generation of specific CTLs in vitro. These CTLs could be important reagents for use in cellular immunotherapy and/or in isolating tumor antigens for potential tumor vaccine development. Int. J. Cancer 78:685–694, 1998. © 1998 Wiley-Liss, Inc.     

The Novel C24D Synthetic Polypeptide Inhibits Binding of Placenta Immunosuppressive Ferritin to Human T Cells and Elicits Anti–Breast Cancer Immunity In Vitro and In Vivo

Immune tolerance mechanisms supporting normal human pregnancy are exploited by breast cancer and other malignancies. We cloned from human placenta and breast cancer cells the novel human immunomodulator named placenta immunosuppressive ferritin (PLIF). PLIF is composed of a ferritin heavy chain–like domain and a novel cytokine-like domain, named C48. Both intact PLIF and C48 inhibit T cell proliferation. Blocking PLIF by specific antibodies in a tolerant breast cancer model in nude mice resulted in tumor cell apoptosis and rejection. This prompted us to study active immune preventive strategies targeting PLIF activity. Currently, we report on the design and synthesis of the novel C24D polypeptide, which inhibits the binding of PLIF to T cells and therefore inhibits the immune suppressive effect of PLIF. The effect of C24D on the generation of anti–breast cancer cytotoxic T lymphocytes (CTLs) was studied in vitro in cultures of MCF-7 (HLA-A2⁺) or T47D (HLA-A2⁻) breast cancer cells incubated with peripheral blood mononuclear cells (PBMCs) from healthy blood donors. We found that C24D treatment exclusively induced development of CTLs. On reactivation by their specific target cells, the CTLs secreted interferon-γ and induced target apoptosis. Anti–MCF-7 CTLs were cross-cytotoxic to MDA-MB-231 (HLA-A2⁺) triple-negative breast cancer but not to T47D. Moreover, C24D treatment in vivo inhibited the growth of MCF-7 tumors engrafted in immune-compromised nude mice transfused with naïve allogeneic human PBMCs. Our results demonstrate that C24D treatment breakdown breast cancer induced tolerance enabling the initiation of effective anti-tumor immune response.Abbreviations: CTLs, cytotoxic T lymphocytes; E:T, effector/target; FITC, fluorescein isothiocyanate; IFN-γ, interferon-γ; IL, interleukin; mAb, monoclonal antibody; PBMCs, peripheral blood mononuclear cells; PBS, phosphate-buffered saline; PLF, placental type isoferritin; PLIF, placenta immunosuppressive ferritin; TNBC, triple-negative breast cancer; TNF-α, tumor necrosis factor–α